Assay for detection of pathogenic leptospira strains

ABSTRACT

The present invention provides methods and compositions for determining the presence and/or amount of pathogenic Leptospira in a test sample. In particular, substantially purified oligonucleotide primers and probes are described that can be used for qualitatively and quantitatively detecting pathogenic Leptospira nucleic acid in a test sample by amplification methods. The present invention also provides primers and probes for generating and detecting control nucleic acid sequences that provide a convenient method for assessing internal quality control of the Leptospira assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/563,037, which is the U.S. National Stage of PCT/US2016/025817, filedApr. 4, 2016, which claims priority from U.S. Provisional ApplicationNo. 62/142,723, filed Apr. 3, 2015.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 15, 2021, isnamed sequence.txt and is 9,333 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods fordetecting pathogenic Leptospira in a test sample.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Leptospirosis is caused by a waterborne spirochete of the genusLeptospira. Until recently, Leptospira species were grouped byserological data into two species, Leptospira interrogans and thenon-pathogenic L. biflexa, together encompassing over 230 serovars. Morerecently, sequence information has allowed Leptospira to be grouped into16 genomospecies, including L. interrogans, biflexa, kirshneri, andborgpetersenii. Unfortunately, the species cannot be neatly categorizedinto pathogenic and non-pathogenic, since both kinds of serovars arepresent in any given genomospecies. Despite this complication, L.interrogans serovars icterohaemorrhagiae, copenhageni, lai, australis,and autumnalis are among those most commonly found in humans, withicterohaemorrhagiae usually causing the most severe symptoms.

The source of Leptospira infection is through exposure to the urine ofan infected animal, although direct contact is not necessary. Infectionis often discovered in patients who have been in contact withcontaminated bodies of water. Leptospira enters the body via cuts andabrasions or by contact with mucosa, incubation lasting from 2-20 days.The bacterium infects first the blood and then CSF, usually beingcleared from both by the third week after symptoms present. Leptospiracan be found in the urine within one week of symptom onset, and maycontinue to be present for months or years without treatment.

The symptoms of leptospirosis have a broad range of severity. Mostinfected individuals are asymptomatic or have very mild symptoms, and donot seek medical attention. Some, however have more severe symptomswhich can lead to death. Symptoms can arise suddenly and include fever,chills, headache, body aches, abdominal pain, conjunctival suffusion,and sometimes a skin rash. The headaches and myalgia may be severe, andup to 25% of patients suffer from aseptic meningitis. Between 5 and 10%of all leptospirosis patients have icteric leptospirosis, sometimescalled Weil's disease, which is a more severe condition that is fatal in5 to 15% of cases. Symptoms include those in the anicteric disease andmay also include jaundice, liver failure or acute renal failure in manycases. Respiratory and cardiac involvement is also common and can leadto respiratory distress syndrome or myocarditis.

The detection of Leptospira in the clinical setting is cumbersome.Serological studies are time consuming and complex, and culture can takefrom 6 to 26 weeks. In addition, the bacterium quickly loses viabilityin urine, the primary sample type, and culture tests provide limitedutility. In contrast, real-time PCR detection is fast, sensitive, anddoes not require organism viability. Samples can be frozen or mixed withpreservative for transport. While the taxonomy of Leptospira is complex,16S sequence data suggests that pathogenic and non-pathogenic subspeciesmay be distinguished by PCR. The disclosed methods and compositions aredesigned to detect pathogenic species only. Non-pathogenic species likeL. biflexa are not detected. In addition, utilizing PCR-based methodswill allow testing of blood, CSF, or urine to give an indication of thestage of infection when tested early.

Several reports disclose assays of patient samples following a nucleicacid amplification step, such as PCR (Brown et al., Evaluation of thepolymerase chain reaction for early diagnosis of leptospirosis. J. Med.Microbiol. 43:110-114, 1995 and Smythe et al., A quantitative PCR(TaqMan) assay for pathogenic Leptospira spp. BMC Infectious Diseases.2(13), 2002), but these references do not teaches a method of detectingonly pathogenic Leptospira DNA. Other relevant references describe thecurrent understanding of the genotypic differences in Leptospiraserovars (Levett, P. N., Leptospirosis. Clinical Microbiology Reviews.14(2):296-36, 2001; Brenner et al., Further determination of DNArelatedness between serogroups and serovars in the family Leptospiraceaewith a proposal for Leptospira alexanderi sp. nov. and four newLeptospira genomospecies. Int. J. Syst. Bacteriol. 49:839-858, 1999;Ramadass et al., Genetic characterization of pathogenic Leptospiraspecies by DNA hybridization. Int. J. Syst. Bacteriol. 42:215-219, 1992;Yasuda et al., Deoxyribonucleic acid relatedness between serogroups andserovars in the family Leptospiraceae with proposals for seven newLeptospira species. Int. J. Syst. Bacteriol. 37:407-415, 1987; WorldHealth Organization. Leptospirosis worldwide, 1999. Wkly. Epidemiol.Rec. WHO 75:217-223, 1999; Edwards and Domm, Human leptospirosis.Medicine 39:117-156, 1960; Kelly, Leptospirosis. p. 1580-1587 from:Gorbach, S. L. et al., Infectious Diseases, 2nd Edition, W.B. Saunders,Philadelphia Pa., 1998).

Yet, in spite of the knowledge in the art, there is not currently amethod for detecting only pathogenic serovars of Leptospira or an assaythat is also capable of distinguishing pathogenic Leptospira from otherspirochetes. The compositions and methods disclosed herein are intendedto provide such a method.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for determiningthe presence and/or amount of pathogenic Leptospira nucleic acids in atest sample. In particular, the invention provides substantiallypurified oligonucleotides for qualitatively and quantitatively detectingLeptospira nucleic acids in a test sample and amplification methods aredescribed herein. The present invention can provide a specific,sensitive method that exhibits a broad dynamic range of detection ofpathogenic Leptospira without detecting unrelated spirochetes ornon-pathogenic serovars, and which can advantageously providequantitative as well as qualitative results. The invention may be usedalone, or in combination with clinical symptoms or other indicators, fordiagnosing an individual as having pathogenic Leptospira.

Accordingly, in one aspect, the disclosure provides oligonucleotideprimers and probes used in the methods described herein to provide anassay for detecting pathogenic Leptospira. In certain embodiments, theinvention provides a substantially purified oligonucleotide having asequence selected from the group consisting of:

(SEQ ID NO: 1) 5′-AGTAACACGTGGGTAATCTTCCT-3′,  (SEQ ID NO: 2)5′-TCTCTCGGGACCATCCAGTA-3′,  and (SEQ ID NO: 3)5′-TGGGATAACTTTCCGAAAGGGAAG C-3′,wherein the oligonuclotide is attached either directly or indirectly toa detectable label.

Direct or indirect attachment can mean that the label can beincorporated into, associated with or conjugated to the oligonucleotide,or the attachment may comprise a spacer arm of various lengths.Attachment may be by covalent or non-covalent means as long as theoligonucleotide is detectable by the means disclosed herein and known inthe art.

The detectable label may be a fluorescent dye or the detectable labelmay comprise a reporter dye and a quencher. In some embodiments, theoligonucleotide of the invention may be

(SEQ ID NO: 3) 5′ [6~FAM]-TGGGATAACTTTCCGAAAGGGAAGC -[BHQ-1] 3′.

In some embodiments, the invention provides a pair of substantially pureoligonucleotide primers comprising SEQ ID NO: 1 and SEQ ID NO: 2. Theprimers may be detectably labeled and they may be used in conjunctionwith a detectably labeled probe. The primer pair can be suitable foramplifying the 16S gene of pathogenic Leptospira or a fragment orcomplement thereof including, but not limited to, SEQ ID NO: 4. The 16Sgene sequences of numerous pathogenic Leptospira serovars are known inthat art, and in some embodiments, the invention provides for primerpairs that are suitable for amplifying the 16S gene sequences ofpathogenic Leptospira serovars, but which do not comprise SEQ ID NO: 1or 2.

In one aspect, the invention provides a detection method for identifyingthe presence or absence of pathogenic Leptospira in a test sample,comprising detecting the presence or absence of a 16S target nucleicacid comprising at least 15 contiguous nucleotides that are at least 95%identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, or a fragment or complement thereof,wherein the presence of said 16S target nucleic acid identifies thepresence of pathogenic Leptospira.

In some embodiments, the detection method further comprises: (a)providing a primer pair suitable for amplifying the 16S target nucleicacid or a fragment thereof, and providing a detectably labeled probesuitable for hybridizing to the 16S target nucleic acid or a fragmentthereof, (b) performing a primer extension reaction comprising theprimer pair of step (a) under conditions suitable to produce a firstreaction product when the 16S target nucleic acid is present in saidsample, and (c) determining the presence or absence of pathogenicLeptospira by detecting the presence or absence of the detectable labelof the probe.

In another aspect, the invention provides that at least one member ofthe primer pair used in the detection method comprises SEQ ID NO: 1 orSEQ ID NO: 2. Alternatively, at least one member of the primer pairconsists of SEQ ID NO: 1 or SEQ ID NO: 2. In another aspect, thedetectably labeled probe may comprise SEQ ID NO: 3 or consist of 5′[6˜FAM]-TGGGATAACTTTCCGAAAGGGAAGC-[BHQ-1] 3′ (SEQ ID NO: 3).

In one aspect, the invention provides a method for detecting thepresence or amount of pathogenic Leptospira nucleic acids in a testsample, comprising:

-   -   (a) amplifying pathogenic Leptospira nucleic acids if present in        the sample using a pair of oligonucleotide primers having the        sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2;    -   (b) hybridizing said amplified pathogenic Leptospira nucleic        acids with an oligonucleotide probe having the sequence set        forth in SEQ ID NO: 3 in the presence of an enzyme that cleaves        said probe when said probe hybridizes to said pathogenic        Leptospira nucleic acids; and    -   (c) detecting a signal from said probe, wherein said signal        indicates the presence or amount of pathogenic Leptospira        nucleic acids in said test sample.

In some embodiments, the test sample can be selected from the groupconsisting of serum, blood, plasma, cerebral spinal fluid, synovialfluid, and urine. In some embodiments, the pathogenic Leptospira nucleicacids are extracted from the test sample prior to amplifying the nucleicacids, while in other embodiments, the test sample may be used directly.In some embodiments, the probe may comprise a reporter dye and aquencher, and in some embodiments, the reporter dye can be 6˜FAM and thequencher can be BHQ-1.

In one aspect, the invention provides a method of diagnosing anindividual suspected of having pathogenic Leptospira, comprising:

-   -   (a) obtaining a sample from said individual suspected of having        pathogenic Leptospira,    -   (b) extracting substantially pure nucleic acids from the sample,    -   (c) performing an amplification reaction in the presence of a        detectably labeled probe comprising SEQ ID NO: 3 and a primer        pair comprising SEQ ID NO: 1 and SEQ ID NO: 2, wherein        hybridization of the detectably labeled probe to a corresponding        sequence of the nucleic acids from the sample in the presence of        a polymerizing enzyme will cleave the detectable label from the        probe when the nucleic acids from the sample are amplified by        the primer pair,    -   (d) detecting a signal from the detectable label of the probe,        wherein said signal indicates the presence or amount of        pathogenic Leptospira nucleic acids in the sample, and    -   (e) determining that the individual suspected of having        pathogenic Leptospira has pathogenic Leptospira if the signal is        detected or diagnosing the individual as not having pathogenic        Leptospira if the signal is not detected.

In some embodiments, the amplification reaction may comprise real-timePCR. In some embodiments, the probe may comprise a reporter dye and aquencher, and in some embodiments, the reporter dye can be 6˜FAM and thequencher can be BHQ-1.

In one aspect, the invention provides a kit comprising a primer pairthat specifically hybridize to a target nucleic comprising SEQ ID NO: 4,a fragment, or a complement thereof, and a probe that specificallyhybridizes the target nucleic acid of SEQ ID NO: 4, a fragment, or acomplement thereof.

In some embodiments of the kit, at least one member of the primer paircomprises the sequence of SEQ ID NO: 1 or 2, or a complement thereof. Insome embodiments, the primer pairs consists a first primer and a secondprimer, wherein the first primer comprises SEQ ID NO: 1, or a complementthereof, and the second primer comprises SEQ ID NO: 2, or a complementthereof. In some embodiments, the probe may comprise SEQ ID NO: 3, or acomplement thereof. In some embodiments, the detectable label on theprobe comprises a reporter dye and a quencher, and in some embodiments,the reporter dye can be 6˜FAM and the quencher can be BHQ-1.

In another aspect, the invention provides a kit comprising a primer pairthat specifically hybridize to a target nucleic acid comprising SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment orcomplement thereof, and a detectably labeled probe that specificallyhybridizes to the target nucleic acid comprising SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment, or a complementthereof. In some embodiments, the detectable label comprises a reporterdye and a quencher, and in some embodiments, the reporter dye can be6˜FAM and the quencher can be BHQ-1.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-1B show typical amplification plots from the disclosedLeptospira assay. FIG. 1A shows high positive, low positive, andnegative results in a linear view. FIG. 1B shows high positive, lowpositive, and negative results in a log view.

FIGS. 2A-2C show the results from the validation of primer and probesets. Set 1 is shown in FIG. 2A, Set 2 is shown in FIG. 2B, and Set 3 isshown in FIG. 2C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for the rapidand sensitive determination of pathogenic Leptospira nucleic acids intest samples. In particular, oligonucleotide probes and primers aredescribed that can be used in methods for quantitatively orqualitatively detecting pathogenic Leptospira nucleic acids in a sample.The present invention also provides primers and probes for generatingand detecting control nucleic acid sequences that provide a convenientmethod for assessing internal quality control of the disclosedLeptospira assay.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to“an oligonucleotide” includes a plurality of oligonucleotide molecules,and a reference to “a nucleic acid” is a reference to one or morenucleic acids.

As used herein, “about” means plus or minus 10%.

As used herein, the term “substantially purified” in reference tooligonucleotides does not require absolute purity. Instead, itrepresents an indication that the sequence is relatively more pure thanin the natural environment. Such oligonucleotides may be obtained by anumber of methods including, for example, laboratory synthesis,restriction enzyme digestion, extraction or isolation from a sample, orPCR. A “substantially purified” oligonucleotide is preferably greaterthan 50% pure, more preferably at least 75% pure, and even morepreferably at least 95% pure, and most preferably 98% pure.

As used herein, the term “oligonucleotides” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides or any combinationthereof. These oligonucleotides are at least 5 nucleotides in length,preferably 10 to 70 nucleotides long, with 15 to 26 nucleotides beingthe most common. In certain embodiments, the oligonucleotides are joinedtogether with or linked to a detectable label.

Oligonucleotides used as primers or probes for specifically amplifying(i.e., amplifying a particular target nucleic acid sequence) orspecifically detecting (i.e., detecting a particular target nucleic acidsequence) a target nucleic acid generally are capable of specificallyhybridizing to the target nucleic acid.

As used herein, the term “suitable for amplifying,” when referring tooligonucleotide primer or primer pairs, is meant primers thatspecifically hybridize to a target nucleic acid and are capable ofproviding an initiation site for a primer extension reaction in which acomplementary copy of the target nucleic acid is synthesized.

As used herein, the term “hybridize” refers to process that twocomplementary nucleic acid strands anneal to each other underappropriately stringent conditions. Hybridizations are typically andpreferably conducted with probe-length nucleic acid molecules,preferably 10-100 nucleotides in length. Nucleic acid hybridizationtechniques are well known in the art. See, e.g., Sambrook, et al., 1989,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Press, Plainview, N.Y. Those skilled in the art understand how toestimate and adjust the stringency of hybridization conditions such thatsequences having at least a desired level of complementarity will stablyhybridize, while those having lower complementarity will not. Forexamples of hybridization conditions and parameters, see, e.g.,Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. etal. 1994, Current Protocols in Molecular Biology. John Wiley & Sons,Secaucus, N.J.

The term “stringent hybridization conditions” as used herein refers tohybridization conditions at least as stringent as the following:hybridization in 50% formamide, 5×SSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS,0.1 mg/mL sonicated salmon sperm DNA, and 5× Denhart's solution at 42°C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washing with0.2×SSC, 0.1% SDS at 45° C. In another example, stringent hybridizationconditions should not allow for hybridization of two nucleic acids whichdiffer over a stretch of 20 contiguous nucleotides by more than twobases.

The terms “target nucleic acid” or “target sequence” as used hereinrefer to a sequence which includes a segment of nucleotides of interestto be amplified and/or detected. Copies of the target sequence which aregenerated during the amplification reaction are referred to asamplification products or amplicons. Target nucleic acids may becomposed of segments of a chromosome, a complete gene with or withoutintergenic sequence, segments or portions of a gene with or withoutintergenic sequence, or sequence of nucleic acids which probes orprimers are designed. Target nucleic acids may include a wild-typesequence(s), a mutation, deletion or duplication, tandem repeat regions,a gene of interest, a region of a gene of interest or any upstream ordownstream region thereof. Target nucleic acids may representalternative sequences or alleles of a particular gene. Target nucleicacids may be derived from genomic DNA, cDNA, or RNA. As used hereintarget nucleic acid may be DNA or RNA extracted from a cell or a nucleicacid copied or amplified therefrom, or may include extracted nucleicacids further converted using a bisulfite reaction.

As used herein, the term “Leptospira nucleic acids” refers to DNA and/orRNA comprising a contiguous sequence from a Leptospira genome, or thecomplement thereof. Leptospira nucleic acids may be Leptospira genomicDNA, Leptospira messenger RNA, or the complement of these sources,obtained by any method including obtaining the nucleic acid from abiological source, synthesizing the nucleic acid in vitro, or amplifyingthe nucleic acid by any method known in the art.

The terms “amplification” or “amplify” as used herein includes methodsfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. A target nucleic acid may be either DNA or RNA.The sequences amplified in this manner form an “amplicon” or“amplification product.” While the exemplary methods describedhereinafter generally relate to amplification using the polymerase chainreaction (PCR), numerous other methods are known in the art foramplification of nucleic acids (e.g., isothermal methods, rolling circlemethods, etc.). The skilled artisan will understand that these othermethods may be used either in place of, or together with, PCR methods.See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Inniset al., Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharam,et al., Nucleic Acids Res. 2001 Jun. 1; 29(11):E54-E54; Hafner, et al.,Biotechniques 2001 April; 30(4):852-6, 858, 860; Zhong, et al.,Biotechniques 2001 April; 30(4):852-6, 858, 860.

The term “complement” “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) refersto standard Watson/Crick pairing rules. The complement of a nucleic acidsequence such that the 5′ end of one sequence is paired with the 3′ endof the other, is in “antiparallel association.” For example, thesequence “5′-A-G-T-3′” is complementary to the sequence “3′-T-C-A-S′.”Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids described herein; these include, forexample, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA). Complementarity need not be perfect; stableduplexes may contain mismatched base pairs, degenerative, or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. A complement sequence can also be asequence of RNA complementary to the DNA sequence or its complementsequence, and can also be a cDNA. The term “substantially complementary”as used herein means that two sequences specifically hybridize (definedabove). The skilled artisan will understand that substantiallycomplementary sequences need not hybridize along their entire length.

As used herein, the term “sample,” “test sample,” or “biological sample”refers to any liquid or solid material believed to comprise Leptospiranucleic acids. In preferred embodiments, a test sample is obtained froma biological source, such as cells in culture or a tissue or fluidsample from an animal, most preferably, a human. Preferred samples ofthe invention include, but are not limited to, plasma, serum, wholeblood, blood cells, lymphatic fluid, cerebrospinal fluid, synovialfluid, urine, saliva, and skin or other organs (e.g. biopsy material).The term “patient sample” as used herein may also refer to a tissuesample obtained from a human seeking diagnosis or treatment of a diseaserelated to a Leptospira infection. Each of these terms may be usedinterchangeably.

The term “detectable label” as used herein refers to a composition ormoiety that is detectable by spectroscopic, photochemical, biochemical,immunochemical, electromagnetic, radiochemical, or chemical means suchas fluorescence, chemifluoresence, or chemiluminescence, or any otherappropriate means. Preferred detectable labels are fluorescent dyemolecules, or fluorochromes, such fluorescein, phycoerythrin, CY3, CY5,allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, FAM, 6˜FAM,JOE, TAMRA, tandem conjugates such as phycoerythrin-CY5, and the like.These examples are not meant to be limiting.

The term “fluorochrome” as used herein refers to a molecule that absorbsa quantum of electromagnetic radiation at one wavelength, and emits oneor more photons at a different, typically longer, wavelength inresponse. In preferred embodiments, a fluorochrome can be a member of apair of physically linked fluorochromes that exhibit fluorescence energytransfer. An energy transfer pair may be excited by a quantum ofelectromagnetic radiation at a wavelength at which the donorfluorochrome is excited; however, fluorescence from the donorfluorochrome that would be expected in the absence of the acceptor isquenched at least in part, and emission at an emission wavelength of theacceptor fluorochrome is observed.

In particularly preferred embodiments, a fluorochrome is one member of aphysically linked “molecular beacon” pair. In these embodiments, themolecular beacon pair may be excited by a quantum of electromagneticradiation at a wavelength at which a first fluorochrome member of thepair is excited; however, fluorescence from the first fluorochrome thatwould be expected in the absence of the second fluorochrome is quenchedat least in part. Unlike energy transfer pairs, however, emission at anemission wavelength of the acceptor fluorochrome is not observed. Thus,these labels comprise a pair of dyes, one of which is referred to as a“reporter,” and the second of which is referred to as a “quencher.” Whenthe two dyes are held in close proximity, such as at the ends of anucleic acid probe, the quencher moiety prevents detection of afluorescent signal from the reporter moiety. When the two dyes areseparated, however, the fluorescent signal from the reporter moietybecomes detectable.

As used herein, “Scorpion primer” or “Scorpion probe” refers to anoligonucleotide having a 3′ primer with a 5′ extended probe tail havinga hairpin structure which possesses a fluorophore/quencher pair.Optionally, the Scorpion primer/probe further contains an amplificationblocker (e.g., hexethylene glycol (“BEG”) separating the probe moietyfrom the primer moiety.

As used herein, the term “Scorpion detection system” refers to a methodfor real-time PCR. This method utilizes a bi-functional molecule(referred to herein as a “Scorpion”), which contains a PCR primerelement covalently linked by a polymerase-blocking group to a probeelement. Additionally, each Scorpion molecule contains a fluorophorethat interacts with a quencher to reduce the background fluorescence.

As used herein, the term “detecting” used in context of detecting asignal from a detectable label to indicate the presence of a targetnucleic acid in the sample does not require the method to provide 100%sensitivity and/or 100% specificity. As is well known, “sensitivity” isthe probability that a test is positive, given that the person has atarget nucleic acid sequence, while “specificity” is the probabilitythat a test is negative, given that the person does not have the targetnucleic acid sequence. A sensitivity of at least 50% is preferred,although sensitivities of at least 60%, at least 70%, at least 80%, atleast 90% and at least 99% are clearly more preferred. A specificity ofat least 50% is preferred, although sensitivities of at least 60%, atleast 70%, at least 80%, at least 90% and at least 99% are clearly morepreferred. Detecting also encompasses assays with false positives andfalse negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% oreven higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or evenhigher.

As used herein “TaqMan® PCR detection” refers to a method for real timePCR. In this method, a TaqMan® probe which hybridizes to the nucleicacid segment amplified is included in the PCR reaction mix. The TaqMan®probe comprises a reporter dye and a quencher fluorophore on either endof the probe and in close enough proximity to each other so that thefluorescence of the reporter is taken up by the quencher. However, whenthe probe hybridizes to the amplified segment, the 5′-exonucleaseactivity of the Taq polymerase cleaves the probe thereby allowing thereporter fluorophore to emit fluorescence which can be detected.

A “fragment” in the context of a gene fragment refers to a sequence ofnucleotide residues which are at least about 20 nucleotides, at leastabout 25 nucleotides, at least about 30 nucleotides, at least about 40nucleotides, at least about 50 nucleotides, or at least about 100nucleotides. The fragment is typically less than about 400 nucleotides,less than about 300 nucleotides, less than about 250 nucleotides, lessthan about 200 nucleotides, or less than 150 nucleotides. In certainembodiments, the fragments can be used in various hybridizationprocedures or microarray procedures to identify specific pathogens.

By “isolated”, when referring to a nucleic acid (e.g., anoligonucleotide) is meant a nucleic acid that is apart from asubstantial portion of the genome in which it naturally occurs. Forexample, any nucleic acid that has been produced synthetically (e.g., byserial base condensation) is considered to be isolated. Likewise,nucleic acids that are recombinantly expressed, produced by a primerextension reaction (e.g., PCR), or otherwise excised from a genome arealso considered to be isolated.

The term “linker” as used herein refers to one or more chemical bonds ora chemical group used to link one moiety to another, serving as adivalent bridge, where it provides a group between two other chemicalmoieties.

Leptospira Assay:

The compositions and methods disclosed herein comprise primers andprobes for the amplification and detection of a target DNA sequence fromLeptospira. The disclosed DNA primers hybridize to flanking targetregions within the 16S ribosomal RNA gene of pathogenic strains ofLeptospira such as L. interrogans and L. kirchneri. The DNA primers usedin this assay do not hybridize to the 16S gene of non-pathogenic strainssuch as L. biflexa, or to strains of intermediate pathogenicity like L.illini. The disclosed compositions and methods will also allow detectionof pathogenic strains only common in particular regions of the world, L.santarosai, and L. weilii.

In some embodiments, the methods comprise obtaining a biological samplefrom a subject, extracting DNA from the sample, and performing adetection assay on the extracted DNA in which the DNA is contacted witha detectably labeled probe and primers that are specific for the 16Sribosomal RNA gene of pathogenic strains of Leptospira.

Sample Collection and Preparation:

Samples may comprise blood, plasma, urine, saliva, cerebral spinal fluid(CSF), tissue samples, or other commonly utilized types of biologicalsample.

In some embodiments, urine may be collected in a sterile, plasticcontainer with a leak-proof cap and then frozen at −20 to −70° C.Repeated freezing and thawing should be avoided. Urine samples may betransported frozen, or, alternatively, immediately transferred into aspecific urine transport tube, such as an Aptima® Urine SpecimenTransport Tube.

In some embodiments, CSF may be collected in a sterile, plasticcontainer with a leak-proof cap. Alternatively, blood may be collectedin sterile tubes, preferably containing EDTA or another anticoagulant.These samples may be stored and transported while refrigerated at 2-8°C.

Nucleic Acid Isolation or Extraction:

The nucleic acid (DNA or RNA) may be isolated from a sample according toany methods well known to those of skill in the art. If necessary, thesample may be collected or concentrated by centrifugation and the like.The cells of the sample may be subjected to lysis, such as by treatmentswith enzymes, heat, surfactants, ultrasonication, or a combinationthereof. The lysis treatment is performed in order to obtain asufficient amount of nucleic acids derived from the pathogens, ifpresent in the sample, to detect using polymerase chain reaction. DNAextraction methods may include, but are not limited to, ethanolprecipitation, organic extraction such as phenol-chloroform extraction,salting out or salt precipitation, cesium chloride density gradients,anion-exchange methods, silica-based methods including commerciallyavailable column kits, and automated high-throughput purificationsystems.

In one embodiment, DNA extraction may be performed using a MagNA Pure LCautomated nucleic acid extraction system or a similar automated nucleicacid extraction system. Numerous commercial kits also yield suitable DNAincluding, but not limited to, QIAamp™ mini blood kit, AgencourtGenfind™, Roche Cobas® Roche MagNA Pure® or phenol-chloroform extractionusing Eppendorf Phase Lock Gels®.

Leptospira Specific Primers and Probes:

In various embodiments of the present invention, oligonucleotide primersand probes can be used in the methods described herein to amplify anddetect target sequences of pathogenic Leptospira. In certainembodiments, target nucleic acids may include the 16S ribosomal RNA geneof pathogenic strains of Leptospira such as L. interrogans and L.kirchneri. In addition, a second set of primers can also be used toamplify one or more control nucleic acid sequences. The target nucleicacids described herein may be detected singly or in a multiplex format,utilizing individual labels for each target.

The skilled artisan is capable of designing and preparing primers thatare appropriate for amplifying a target sequence in view of thisdisclosure. The length of the amplification primers for use in thepresent invention depends on several factors including the nucleotidesequence identity and the temperature at which these nucleic acids arehybridized or used during in vitro nucleic acid amplification. Theconsiderations necessary to determine a preferred length for anamplification primer of a particular sequence identity are well known tothe person of ordinary skill in the art.

In a preferred embodiment, the Leptospira specific primers are5′-AGTAACACGTGGGTAATCTTCCT-3′ (SEQ ID NO: 1) and5′-TCTCTCGGGACCATCCAGTA-3′ (SEQ ID NO: 2), although the skilled artisanwill understand that other probes may be used. Alternative primers maybe 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1or SEQ ID NO: 2. In other embodiments, primers may be designed such thatthey bind to and are suitable for amplifying a target sequence of theLeptospira 16S gene. For instance, primers may be designed to amplifySEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, or fragments or complements thereof.

Primers may be between 10 and 30 nucleotides long. For instance, primersmay be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides in length. One of skill in the artwill understand that the precise length and composition of the primer isdependent on the sample and the assay conditions. As disclosed herein,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, or fragments or complements thereof provide suitabletarget sequences for designing primers capable of detecting pathogenicLeptospira. In some embodiments primers comprising 10-30 nucleotides ofSEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, or fragments or complements thereof may be used for thedetection of pathogenic Leptospira. In other embodiments, primers thatare at least 90%, at least 95%, or at least 99% identical to sequencescomprising 10-30 nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments orcomplements thereof may be used for the detection of pathogenicLeptospira. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 SEQ ID NO: 11, SEQID NO: 12, and SEQ ID NO: 13 are shown in Table 1.

TABLE 1  16S Sequences SEQ ID NO Sequence 45′-AACTAACGCTGGCGGCGCGTCTTAAACATGCAAGTCAAGCGGAGTAGCAATACTCAGCGGCGAACGGGTGAGTAACACGTGGGTAATCTTCCTCCGAGTCTGGGATAACTTTCCGAAAGGGGAGCTAATACTGGATGGTCCCGAGAGAGGTCATATGATTTTTCGGGTAAAGATTTATTGCTCGGAGCTGAGCCCGCGCCCGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCGGTAGCCGGCCTGAGAGGGTGTTCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTTAAGAATCTTGCTCAATGGGGGGAACCCTGAAGCAGCGACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCAATAAGCAGGGAAAAATAAGCAGCGATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTGTTCGGAATCATTGGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGAAAACTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTGGAGTTTGGGAGAGGCAAGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGCCTAAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTAGATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCAGTTGTTGGGGGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACCGCCTGGGGACTATGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAAAAACCTCACCTAGGCTTGACATGGAGTGGAATTATGTAGAGATACATGAGCCTTCGGGCCGCTTCACAGGTGCTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCACCTTATGTTGCCATCATTTAGTTGGGCACTCGTAAGGAACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCCTCATGGCCTTTATGTCTAGGGCAACACACGTGCTACAATGGCCGGTACAAAGGGTAGCCAACTCGCGAGGGGGAGCTAATCTCAAAAAGCCGGTCCCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT-3′ 55′-AACTAACGCTGGCGGCGCGTCTTAAACATGCAAGTCAAGCGGAGTAGCAATACTCAGCGGCGAACGGGTGAGTAACACGTGGGTAATCTTCCTCCGAGTCTGGGATAACTTTCCGAAAGGGGAGCTAATACTGGATGGTCCCGAGAGAGGTCATATGATTTTTCGGGTAAAGATTTATTGCTCGGAGCTGAG-3′ 65′-GTGTTCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTTAAGAATCTTGCTCAATGGGGGGAACCCTGAAGCAGCGACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCAATAAGCAGGGAAAAATAAGCAGCGATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTGTTCGGAATCATTGGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGAAAACTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTGGAGTTTGGGAGAGGCAAGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGCCTAAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTAGATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCAGTTGTTGGGGGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACCGCCTGGGGACTATGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAAAAACCTCACCTAGGCTTGACATGGAGTGGAATTATGTAGAGATACATGAGCCTTCGGGCCGCTTCACAGGTGCTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCACCTTATGTTGCCATCATTTAGTTGGGCACTCGTAAGGAACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCCTCATGGCCTTTATGTCTAGGGCAACACACGTGCTACAATGGCCGGTACAAAGGGTAGCCAACTCGCGAGGGGGAGCTAATCTCAAAAAGCCGGTCCCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT-3′ 115′-AACTAACGCTGGCGGCGCGTCTTAAACATGCAAGTCAAGCGGAGTAGCAATACTCAGCGGCGAACGGGTGAGTAACACGTGGGTAATCTTCCTCTGAGTCTGGGATAACTTTCCGAAAGGGAAGCTAATACTGGATGGTCCCGAGAGATCATAAGATTTTTCGGGTAAAGATTTATTGCTCGGAGATGAGCCCGCGTCCGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCGGTAGCCGGCCTGAGAGGGTGTTCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTTAAGAATCTTGCTCAATGGGGGGAACCCTGAAGCAGCGACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCAGTAAGCAGGGAAAAATAAGCAGCAATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTGTTCGGAATCATTGGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGAAAACTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTGGAGTTTGGGAGAGGCAAGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGCCTAAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTAGATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCAGTTGTTGGGGGGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACCGCCTGGGGACTATGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAAAAACCTCACCTAGGCTTGACATGGAGTGGAATCATGTAGAGATACATGAGCCTTCGGGCCGCTTCACAGGTGCTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCACCTTATGTTGCCATCATTCAGTTGGGCACTCGTAAGGAACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCCTCATGGCCTTTATGTCTAGGGCAACACACGTGCTACAATGGCCGGTACAAAGGGTAGCCAACTCGCGAGGGGGAGCTAATCTCAAAAATCCGGTCCCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT-3′ 125′-AACTAACGCTGGCGGCGCGTCTTAAACATGCAAGTCAAGCGGAGTAGCAATACTCAGCGGCGAACGGGTGAGTAACACGTGGGTAATCTTCCTCTGAGTCTGGGATAACTTTCCGAAAGGGAAGCTAATACTGGATGGTCCCGAGAGATCATAAGATTTTTCGGGTAAAGATTTATTGCTCGGAGATGAG-3′ 135′-GTGTTCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTTAAGAATCTTGCTCAATGGGGGGAACCCTGAAGCAGCGACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCAGTAAGCAGGGAAAAATAAGCAGCAATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTGTTCGGAATCATTGGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGAAAACTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTGGAGTTTGGGAGAGGCAAGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGCCTAAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTAGATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCAGTTGTTGGGGGGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACCGCCTGGGGACTATGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAAAAACCTCACCTAGGCTTGACATGGAGTGGAATCATGTAGAGATACATGAGCCTTCGGGCCGCTTCACAGGTGCTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCACCTTATGTTGCCATCATTCAGTTGGGCACTCGTAAGGAACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCCTCATGGCCTTTATGTCTAGGGCAACACACGTGCTACAATGGCCGGTACAAAGGGTAGCCAACTCGCGAGGGGGAGCTAATCTCAAAAATCCGGTCCCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT-3′

Additional primers that amplify a target nucleic acid sequence can bedesigned using, for example, a computer program such as OLIGO (MolecularBiology Insights, Inc., Cascade, CO). Important features when designingoligonucleotides to be used as amplification primers include, but arenot limited to, an appropriate size amplification product to facilitatedetection (e.g., by electrophoresis or real-time PCR), similar meltingtemperatures for the members of a pair of primers, and the length ofeach primer (i.e., the primers need to be long enough to anneal withsequence-specificity and to initiate synthesis but not so long thatfidelity is reduced during oligonucleotide synthesis). Typically,oligonucleotide primers are 15 to 35 nucleotides in length.

In some embodiments, a mix of primers can be provided having degeneracyat one or more nucleotide positions. Degenerate primers are used in PCRwhere variability exists in the target sequence, i.e. the sequenceinformation is ambiguous. Typically, degenerate primers will exhibitvariability at no more than about 4, no more than about 3, preferably nomore than about 2, and most preferably, no more than about 1 nucleotideposition.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers. As witholigonucleotide primers, oligonucleotide probes usually have similarmelting temperatures, and the length of each probe must be sufficientfor sequence-specific hybridization to occur but not so long thatfidelity is reduced during synthesis. Oligonucleotide probes aregenerally 15 to 60 nucleotides in length.

In a preferred embodiment, the Leptospira specific probe is 5′[6˜FAM]-TGGGATAACTTTCCGAAAGGGAAGC-[BHQ1] 3′ (SEQ ID NO: 3), although theskilled artisan will understand that other probes may be used.Alternative hybridization probes may be 70, 75, 80, 85, 90, 95, 96, 97,98, or 99% identical to SEQ ID NO: 3. In other embodiments, probes maybe designed such that they hybridize to and are suitable for detecting atarget sequence of the Leptospira 16S gene. For instance, probes may bedesigned to hybridize to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments or complementsthereof.

Probes may be between 10 and 30 nucleotides long. For instance, probesmay be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides in length. One of skill in the artwill understand that the precise length and composition of a probe isdependent on the sample and the assay conditions. As disclosed herein,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, or fragments or complements thereof provide suitabletarget sequences for designing probes capable of detecting pathogenicLeptospira. In some embodiments a probe comprising 10-30 nucleotides ofSEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, or fragments or complements thereof may be used for thedetection of pathogenic Leptospira. In other embodiments, a probe thatis at least 90%, at least 95%, or at least 99% identical to sequencescomprising 10-30 nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments orcomplements thereof may be used for the detection of pathogenicLeptospira. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQID NO: 12, and SEQ ID NO: 13 are shown in Table 1 above.

Additional probes that can detect a target nucleic acid sequence can bedesigned using, for example, a computer program such as OLIGO (MolecularBiology Insights, Inc., Cascade, CO). Important features when designingoligonucleotides to be used as probes include, but are not limited to,an appropriate size target sequence to facilitate detection (e.g., byelectrophoresis or real-time PCR), similar or higher meltingtemperatures compared to reaction primers (if being used in anamplification reaction like RT-PCR), and the length of the probe (i.e.,the probe needs to be long enough to anneal with sequence-specificitybut not so long that fidelity is reduced). Typically, oligonucleotideprobes are 5 to 40 nucleotides in length.

Amplification and Detection of a Target Sequence:

Nucleic acid samples or isolated nucleic acids may be amplified byvarious methods known to the skilled artisan. Preferably, PCR is used toamplify nucleic acids of interest. Briefly, in PCR, two primer sequencesare prepared that are complementary to regions on opposite complementarystrands of a target sequence. An excess of deoxynucleotide triphosphates(dNTPs) are added to a reaction mixture along with a DNA polymerase,e.g., Taq polymerase.

If the target sequence is present in a sample, the primers will bind tothe sequence and the polymerase will cause the primers to be extendedalong the target sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the marker to form reaction products, excessprimers will bind to the marker and to the reaction products and theprocess is repeated, thereby generating amplification products. Cyclingparameters can be varied, depending on the length of the amplificationproducts to be extended. An internal positive amplification control(IPC) can be included in the sample, utilizing oligonucleotide primersand/or probes. The IPC can be used to monitor both the conversionprocess and any subsequent amplification.

In some embodiments, the PCR reaction may comprise thermocycling of 35,40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or up to 60 cycles. Thedenaturing temperature may range from about 95 to 105° C. or about 95°C., and the elongation temperature may range from about 50 to 75° C. orabout 60° C. The cycling may be set up such that the denaturingtemperature is maintained from 1 second to 3 minutes. The cycling may beset up such that the elongation temperature is maintained from 1 secondto 3 minutes. One of skill in the art will know that these parametersmay be optimized depending on, among other things, the samples andprimers being used.

Real time PCR is performed using any suitable instrument capable ofdetecting the accumulation of the PCR amplification product. Mostcommonly, the instrument is capable of detecting fluorescence from oneor more fluorescent labels. For example, real time detection on theinstrument (e.g. an ABI Real-Time PCR System 7500® sequence detector)monitors fluorescence and calculates the measure of reporter signal, orRn value, during each PCR cycle. The threshold cycle, or Ct value, isthe cycle at which fluorescence intersects the threshold value. Thethreshold value can be determined by the sequence detection systemsoftware or manually.

Amplification of nucleic acids can be detected by any of a number ofmethods well-known in the art such as gel electrophoresis, columnchromatography, hybridization with a probe, sequencing, melting curveanalysis, or “real-time” detection. For real-time detection, primersand/or probes may be detectably labeled to allow differences influorescence when the primers become incorporated or when the probes arehybridized, for example, and amplified in an instrument capable ofmonitoring the change in fluorescence during the reaction. Real-timedetection methods for nucleic acid amplification are well known andinclude, for example, the TaqMan® system, Scorpion™ primer system anduse of intercalating dyes for double stranded nucleic acid.

In some embodiments, amplified nucleic acids are detected byhybridization with a specific probe. Probe oligonucleotides,complementary to a portion of the amplified target sequence may be usedto detect amplified fragments. Hybridization may be detected in realtime or in non-real time. Amplified nucleic acids for each of the targetsequences may be detected simultaneously (i.e., in the same reactionvessel) or individually (i.e., in separate reaction vessels). Forsequence-modified nucleic acids, the target may be independentlyselected from the top strand or the bottom strand. Thus, all targets tobe detected may comprise top strand, bottom strand, or a combination oftop strand and bottom strand targets.

In some embodiments, the Leptospira specific primers can be used in apolymerase chain reaction to amplify and detect pathogenic Leptospira.The PCR reaction may comprise sterile nuclease free water, forward andreverse primers, Leptospira specific probe(s), internal control primers,and a “master mix” comprising a DNA polymerase, dNTPs, salts, reactionbuffer, and magnesium. Forward and reverse primers may be used at areaction concentration ranging from 100 nM to 1.5 μM, or moreparticularly from 250-950 nM, 350-850 nM, 450-750 nM, 550-650 nM. Insome embodiments, the forward and reverse primers may be used at areaction concentration of 450, 460, 470, 480, 490, 500, 510, 520, 530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, or 850 nM. Leptospira specific probes may be used at areaction concentration ranging from 1-500 nM, or more particularly, from25-400 nM, 50-300 nM, or 75-200 nM. In some embodiments, the Leptospiraspecific probe may be used at a reaction concentration of 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, or 250 nM.

In some embodiments, the PCR master mix may contain a DNA polymerase,salts, magnesium, dNTPs, and reaction buffer, among other necessary andoptional constituents.

TaqMan® probes (Heid, et al., Genome Res 6: 986-994, 1996) use thefluorogenic 5′ exonuclease activity of Taq polymerase to measure theamount of target sequences in test samples. TaqMan® probes areoligonucleotides that contain a reporter dye usually at or near the 5′base, and a quenching moiety typically at or near the 3′ base. Thequencher moiety may be a dye, such as TAMRA, a Black Hole Quencher, orit may be a non-fluorescent molecule such as4-(4-dimethylaminophenylazo) benzoic acid (DABCYL). See Tyagi, et al.,16 Nature Biotechnology 49-53 (1998). When irradiated, the excitedfluorescent reporter transfers energy to the nearby quenching moiety byFRET rather than fluorescing. Thus, the close proximity of the reporterand quencher prevents emission of donor fluorescence while the probe isintact.

TaqMan® probes are designed to anneal to an internal region of a PCRproduct. When the polymerase replicates a template on which a TaqMan®probe is bound, its 5′ exonuclease activity cleaves the probe. This endsthe activity of the quencher (no FRET) and the reporter fluorophorestarts to emit fluorescence which increases in each cycle proportionalto the rate of probe cleavage. Accumulation of PCR product is detectedby monitoring the increase in fluorescence of the reporter dye (notethat primers may not be labeled). If the quencher is an acceptorfluorophore, then accumulation of PCR product can be detected bymonitoring the decrease in fluorescence of the acceptor fluorophore.

Some embodiments of the disclosed compositions and methods comprise aprobe specific for the Leptospira amplicon. The probe used for detectionof the Leptospira amplicon may optionally be labeled with a reporter dyeand a quencher as with a TaqMan® probe. For instance, an exemplary probemay comprise a FAM reporter dye on its 5′ end and a BHQ-1 quencher onits 3′ end. Those of skill in the art will know that this is only oneexample of numerous reporter day and quencher combinations that may beutilized in such an assay. Useful reporter dyes include, but are notlimited to, BODIPY FL, FAM, 6˜FAM, VIC,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives (acridine, acridine isothiocyanate) Alexa Fluor® 350, AlexaFluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, AlexaFluor® 594, Alexa Fluor® 647 (Molecular Probes),5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamid, OregonGreen 488, Rhodamine green, Oregon Green 514, TET, Cal Gold, BODIPY R6G,Yakima Yellow, JOE, HEX, Cal Orange, BODIPY TMR-X, Quasar-570/Cy3,TAMRA, Rhodamine Red-X, Rhodamine Red, BODIPY 581/591, Cy3.5, ROX, CalRed, Texas Red, BODIPY TR-X, BODIPY 630/665-X, Pulsar-650,Quasar-670/Cy5, and Cy5.5.

Suitable quenchers are selected based on the fluorescence spectrum ofthe particular fluorophore. Useful quenchers include, for example, theBlack Hole™ quenchers BHQ-1, BHQ-2, and BHQ-3 (Biosearch Technologies,Inc.), and the ATTO-series of quenchers (ATTO 540Q, ATTO 580Q, and ATTO612Q; Atto-Tec GmbH). Other useful quenchers include, but are notlimited to, dabcyl, QSY 35, Eclipse, QSY 7, QSY 9, ElleQuencher, Iowablack, QSY 21, Qxl quenchers, Iowa black FQ, Iowa black RQ, and IRDyeQC-1.

One of skill in the art will understand that certain pairing of reporterdyes and quenchers are preferred. For instance, in one embodiment, thereporter/quencher pair is 6˜FAM/BHQ1. In another embodiment, thereporter may be Quasar-670, Cal Red, Quasar-570, or TAMRA and thequencher my BHQ-1, BHQ-2, or BHQ-3.

When the reporter dye and quencher are in close proximity (i.e. both arepresent on an intact oligonucleotide probe) the fluorescence of thereporter is suppressed. However, when the oligonucleotide probe iselongated in the disclosed assay, the 5′-3′ nuclease activity of DNApolymerase will cleave the probe if it is bound specifically to thetarget sequence between the forward and reverse primer sites. Thisreleases the reporter dye and quencher, and upon excitation, thefluorescent signal produced by the reporter dye is no longer quenched.This results in an increase in fluorescence that can be detected.

In some embodiments of the disclosed compositions and methods, BlackHole Quencher may be used to reduce background. In a preferredembodiment, the Letospira specific probe comprises a reporter dye and aquencher so that it can be detected upon amplification of the targetsequence.

Additional, detectable labels include, but are not limited to,radioisotopes (e.g., ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I), electron-densereagents (e.g., gold), enzymes (e.g., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels (e.g., colloidal gold), magnetic labels (e.g., Dynabeads™),biotin, dioxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available. Other labels include ligands oroligonucleotides capable of forming a complex with the correspondingreceptor or oligonucleotide complement, respectively.

The label can be directly incorporated into the nucleic acid to bedetected, or it can be attached to a probe (e.g., an oligonucleotide) orantibody that hybridizes or binds to the nucleic acid to be detected. Alabel can be attached either directly or indirectly to probes orprimers, and a label may be attached by covalent or non-covalent means.A label can be attached by spacer arms of various lengths to reducepotential steric hindrance or impact on other useful or desiredproperties. See, e.g., Mansfield, 9 Mol. Cell. Probes 145-156 (1995).Detectable labels can be incorporated into nucleic acids by covalent ornon-covalent means, e.g., by transcription, such as by random-primerlabeling using Klenow polymerase, or nick translation, or amplification,or equivalent as is known in the art. For example, a nucleotide base isconjugated to a detectable moiety, such as a fluorescent dye, and thenincorporated into nucleic acids during nucleic acid synthesis oramplification.

Alternatively, the detectable labels may be part of a Scorpion detectionsystem. With Scorpion detection systems, sequence-specific priming andPCR product detection is achieved using a single molecule. The Scorpionprobe maintains a stem-loop configuration in the unhybridized state. Thefluorophore is quenched by a moiety coupled to the 5′ end, although insuitable embodiments, the fluorophore is attached to the 5′ end and isquenched by a moiety coupled to the 3′ end. The 3′ portion of the stemalso contains a sequence that is complementary to the extension productof the primer. This sequence is linked to the 5′ end of the Scorpionprobe via a non-amplifiable monomer. After extension using the Scorpionprimer, the specific probe sequence is able to bind to its complementwithin the extended amplicon thus opening up the hairpin loop. Thisprevents fluorescence from being quenched and a signal is observed. Aspecific target is amplified by the reverse primer and the primerportion of the Scorpion, resulting in an extension product. Afluorescent signal is generated due to the separation of the fluorophorefrom the quencher resulting from the binding of the probe element of theScorpion to the extension product.

Internal Controls:

To ensure the absence of non-specific PCR inhibitors in a sample, aninternal positive amplification control (IPC) may be included with eachspecimen. The positive control primers and probe can be added to createa multiplex reaction with the target and sample primers. The IPCamplicon can be detected with a probe labeled with a reporter dyeattached to the 5′ end of the probe. A sample can be interpreted asnegative if the analysis of the internal positive control indicates thatDNA amplification has occurred in the reaction tube but there was nodetection of the Leptospira specific probe.

EXAMPLES Example 1. Sample Preparation and DNA Extraction

One 250 μl aliquot of “Leptospira Positive Control” control was removedand thawed at room temperature. 200 μl sterile water was used for anegative control. 1 positive and 1 negative control were performed foreach batch of extractions. 200 μl of control or specimen was pipettedinto a given well of the sample cartridge. Refrigerated or frozen urinesamples mixed with urine transport media were allowed to warm to roomtemperature (18° to 26° C.) before loading into the sample cartridge.

Internal Positive Control DNA was added to a lysis buffer prior toextraction, and 200 μl was added to each well containing a sample.

Example 2. DNA Amplification and Detection

DNA amplification was carried out using an ABI 7500 Real-Time PCRSystem. Prior to amplification, samples were combined with a master mix.

The amplification plots of the controls were examined. The plots showeda sigmoidal curve with a distinct exponential growth phase followed by aplateau phase.

Sample amplifications were examined as well. Negative plots were onlyvisible as a line near the bottom of the graph, due to poor resolutionat lower fluorescence values.

An example of typical curves for the Leptospira assay are shown in FIGS.1A and 1B. The curve on the left is typical of a high positive result.The curve on the right is typical of a low positive result. Thehorizontal line is the threshold. All positive results cross this line.The cycle at which a given plot crosses the threshold line is called theThreshold Cycle (Ct). Negative plots do not have sigmoidal shape, and/ordo not cross the threshold line. The data may be viewed in a linearformat, as in FIG. 1A, or a log format, as in FIG. 1B. The decision onwhich format to use to visualize amplification plots is left to thediscretion of the technician, however each plot has advantages. Positiveresults may be more intuitively obvious in linear format, since onlypositive plots are distinguishable above the background in this format.Log view, however, allows better discernment of small differencesbetween plot lines, especially at low fluorescence.

If the amplification plots of the controls do not show curves, it may bean indication that there has been contamination, improper preparation ofthe master mix or the controls, or degradation of the fluorescent probe.The IPC must amplify, and it must be within the specified Ct range forthe IPC result to be valid. If the IPC results are out of the specifiedCt range and targets are not detected, then target results may not bevalid.

Examination of clinical samples was done after the controls wereexamined and shown to have the correct results. Amplification plots wereexamined for every sample. When the amplification plot showed anexponential increase the amplification curve was considered a valid,positive result.

Example 3: Validation of Primers and Probes for DistinguishingPathogenic and Non-Pathogenic Leptospira

Leptospira weillii (ATCC #43285) is a non-interrogans pathogen whichmight have trouble being detected in the Leptospira assay. Multiple setsof primers were tested in order to determine effectiveness of detection.

For validation, thawed bacteria from ATCC was diluted 10 times andfrozen in 250 μl or 1 ml aliquots. Thawed samples were extracted forDNA. Samples were roughly estimated to have 10⁸ cells/ml. DNA fromserial dilutions of the bacteria were used to determine the detectionlimits of various primer pairs.

Three different sets of primer pairs were used for validation tests. Set1 consisted of primers F76 and R148, Set 2 consisted of F71 and R148,and Set 3 consisted of FSmythe and RSmythe. The sequences of theseprimers can be found in Table 2. For Sets 1 and 2, probe P101 was usedto detect target sequence amplification, and for Set 3, PSmythe was usedto detect target sequence amplification.

TABLE 2  Sequences of Primers and Probes Name Sequence SEQ ID NO: F765′-CACGTGGGTAATCTTCCTCTG-3′ 7 R148 5′-TCTCTCGGGACCATCCAGTA-3′ 2 F715′-AGTAACACGTGGGTAATCTTCCT-3′ 1 FSmythe 5′-CCCGCGTCCGATTAG-3′ 8 RSmythe5′-TCCATTGTGGCCGR*ACAC-3′ 9 P101 5′-TGGGATAACTTTCCGAAAGGGAAGC-3′ 3PSmythe 5′-CTCACCAAGGCGACGATCGGTAGC-3′ 10 *R indicates a position thatis A 50% of the time and G 50% of the time. Primer lots meeting thiscondition can be bought commercially.

100 μM of each primer in a given set and 10 μM of the correspondingprobe were combined in a reaction mixture comprising master mix, taqpolymerase, Leptospira weillii DNA, and water. Thermocycling was carriedout using an ABI 7500 Real-Time PCR System

The results of the real time detection of amplification are found inTable 3.

TABLE 3 Detection Results of Primer Sets Repli- Repli- Repli- AverageSt. % Cell/ml cate 1 cate 2 cate 3 Ct Dev. CV Primer Set 1 10⁸ 22.6823.68 22.38 22.91 0.681 2.97 10⁷ 26 25.99 25.69 25.89 0.176 0.68 10⁶29.82 28.62 29.35 29.26 0.605 2.07 10⁵ 33.95 31.77 33.28 33 1.117 303810⁴ 37.63 34.6  37.35 36.53 1.674 4.58 Negative 45.92 Unde- 43.2  — — —tected Primer Set 2 10⁸ 21.64 22.39 21.47 21.83 0.49  2.24 10⁷ 24.8925.07 24.5  24.82 0.291 1.17 10⁶ 28.53 28.39 34.07 30.33 3.24  10.68 10⁵32.2 30.64 31.77 31.54 0.806 2.55 10⁴ 35.79 34.71 35.52 35.34 0.562 1.59Negative Unde- Unde- Unde- — — — tected tected tected Primer Set 3 10⁸18.08 17.96 17.97 18 0.067 0.37 10⁷ 21.18 21.29 21.28 21.25 0.061 0.2910⁶ 25.06 24.86 25.07 25 0.118 0.47 10⁵ 28.9 29.12 28.79 28.94 0.1680.58 10⁴ 33.57 32.7  33.27 33.18 0.442 1.33 Negative Unde- Unde- Unde- —— — tected tected tected

The cycling output data can be seen in FIG. 2. The bold horizontal linesrepresent the threshold limit.

The PCR efficiency of Set 1 was 97.8%. The PCR efficiency of Set 2 was99.0%. The PCR efficiency of Set 3 was 91.3%. While the primer setsdiffered in terms of Ct scores, Sets 1 and 2 had significantly higherPCR efficiencies than Set 3. This validation indicates that Sets 1 and 2are more suitable for reliable detection of pathogenic Leptospira.Additionally, Sets 1 and 2 had superior limits of detection than Set 3,indicating that Set 3 would likely have trouble detecting some speciesof Leptospira.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Non-limiting embodiments are set forth within the following claims.

What is claimed is:
 1. A substantially purified oligonucleotide having asequence selected from the group consisting of: (SEQ ID NO: 1)5′-AGT AAC ACG TGG GTA ATC TTC CT-3′, (SEQ ID NO: 2)5′-TCTCTCGGGACCATCCAGTA-3′,  and (SEQ ID NO: 3)5′-TGGGATAACTTTCCGAAAGGGAAG C-3′, 

wherein said oligonuclotide is attached either directly or indirectly toa detectable label.
 2. The oligonucleotide of claim 1, wherein thedetectable label is a fluorescent dye.
 3. The oligonucleotide of claim1, wherein the detectable label comprises a reporter dye and a quencher.4. The oligonucleotide of claim 3, wherein the oligonucleotide is 5′[6˜FAM]-TGGGATAACTTTCCGAAAGGGAAGC-[BHQ-1] 3′ (SEQ ID NO: 3).
 5. A pairof substantially pure oligonucleotide primers comprising SEQ ID NO: 1and SEQ ID NO:
 2. 6. A kit comprising: a primer pair that specificallyhybridize to a nucleic acid having a target 16S sequence of SEQ ID NO: 4or a complement thereof and a detectably labeled probe that specificallyhybridizes to a nucleic acid having the sequence of SEQ ID NO: 4 or acomplement thereof, wherein the at least one member of the primer paircomprises the sequence of SEQ ID NO: 1 or 2, or a complement thereof. 7.The kit of claim 6, wherein the probe comprises the sequence of SEQ IDNO: 3, or a complement thereof.
 8. The kit of claim 6, wherein thedetectable label comprises a reporter dye and a quencher.
 9. The kit ofclaim 8, wherein the reporter dye is 6˜FAM.
 10. The kit of claim 8,wherein the quencher is BHQ-1.
 11. A kit comprising: a primer pair thatspecifically hybridize to a target nucleic acid comprising SEQ ID NO: 5or SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment orcomplement thereof, and a detectably labeled probe that specificallyhybridizes to the target nucleic acid comprising SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment or a complementthereof.
 12. The kit of claim 11, wherein the detectable label comprisesa reporter dye and a quencher.